Published on Web 09/28/2002
The Charge Conduction Properties of DNA Holliday Junctions Depend Critically on the Identity of the Tethered Photooxidant Richard P. Fahlman,† Rajendra D. Sharma,‡ and Dipankar Sen*,†,‡ Contribution from Department of Molecular Biology & Biochemistry and Department of Chemistry, Simon Fraser UniVersity, Burnaby, British Columbia V5A 1S6, Canada Received April 5, 2002
Abstract: The mechanism for electrical charge conduction in DNA has been the subject of much recent interest and debate. Many of the measurements of DNA conductivity have been made in aqueous solution, with an aromatic photooxidant moiety such as anthraquinone or a rhodium(III) complex covalently tethered to the DNA. Such studies, however, have given discrepant results, for instance, regarding the relative ability of AT- and GC-rich sequences to conduct charge and the possibility of thymine cyclobutane dimer repair through the DNA from a distance. A recent paper on conduction in DNA immobile four-way junctions using the rhodium photooxidant reported conduction in all four helical arms, contrary to what is known about the three-dimensional structure and stacking of 4-way junctions. We have reexamined conduction in such junctions using rhodium [Rh(phi)2(byp*)Cl3] as well as the anthraquinone photooxidants, and find that although our rhodium data agree with the previously published work, the anthraquinone data reveal conduction in only two of the four helical arms, consistent with the known tertiary structure of four-way junctions. An electrophoretic investigation revealed the formation of intermolecular aggregates in the rhodiumderivatized junctions, but not in the anthraquinone-labeled junctions. Rhodium-specific aggregation was also observed with simple DNA duplexes under the same experimental conditions. A characteristic property of aggregation was that all participating DNA molecules required the rhodium derivatization, and underivatized molecules did not aggregate with the derivatized ones. It is conceivable that the results reported here will help reconcile the various discrepancies that have been reported from charge conduction experiments carried out on DNA utilizing different photooxidants.
Introduction
The first conjectures on the potential of DNA to conduct electrical charge date back to nearly forty years ago.1-2 However, the technologies appropriate to testing such hypotheses have not been available until the past decade. The first studies reported divergent conclusions about the efficiency, distance dependence, and rates of observed charge transfer through DNA double helices. A number of studies reported that the aromatic base stacks of a DNA duplex act as an efficient electrical conductor;3-4 however, other reports concluded that duplex DNA behaved essentially like an insulator.5-6 Such conflicting observations have stimulated much further study of charge transfer through DNA. To date, the wealth of experi* To whom correspondence should be addressed: Department of Molecular Biology & Biochemistry, Simon Fraser University, 8888 University Way, Burnaby, British Columbia V5A 1S6, Canada. Tel.: 604291-4386. Fax: 604-291-5583. E-mail:
[email protected]. † Department of Molecular Biology & Biochemistry, Simon Fraser University. ‡ Department of Chemistry, Simon Fraser University. (1) Eley, D. D.; Spivey, D. I. Trans. Faraday Soc. 1962, 58, 411-415. (2) Hoffman, T. A.; Ladik, J. AdV. Chem. Phys. 1964, 7, 84-158. (3) Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.; Horrman, A.; Olsen, E. J. C.; Barbara, P. F. Science 1996, 273, 475-480. (4) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlin, N. D.; Bossman, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025-1029. (5) Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. L.; Greenfield, S. R.; Wasielewski, M. R. Science 1997, 277, 673-676. (6) Debije, M. G.; Milano, M. T.; Bernhard, W. A. Angew. Chem., Int. Ed. 1999, 38, 2752-2756. 10.1021/ja020495n CCC: $22.00 © 2002 American Chemical Society
mental data recorded and evaluated have made it evident that, likely, more than a single mechanism exists for charge transfer through DNA. One possible mechanism is a long-range charge “hopping”, whereby an electron hole migrates through the duplex using the oxidizable guanine bases as “steps”7 or by a polaron like hopping mechanism.8 A second prevalent mechanism, for rapid, short-range charge transfer, is the single-step superexchange mechanism, where the DNA behaves essentially as a molecular wire having a continuous molecular orbital.9-10 Beyond the issue of the precise mechanism(s) of charge transfer, a consensus has emerged that continuous base-stacking throughout a DNA duplex is important. Efficiency of charge transfer is observed to be lower in duplexes containing either mismatches11-13 or bulges.14 However, not all perturbations to the helix have prevented charge transfer, as has been noted in helices containing abasic sites15 and short, single-stranded overhangs.16 Nevertheless, even these latter structures have been (7) (8) (9) (10) (11) (12) (13) (14)
Giese, B. Acc. Chem. Res. 2000, 33, 631-636. Schuster, G. B. Acc. Chem. Res. 2000, 33, 253-260. Turro, N. J.; Barton, J. K. J. Biol. Inorg. Chem. 1998, 3, 201-209. Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res. 2001, 34, 159-170. Kelly, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 9861-9870. Giese, B.; Wessely, S. Angew. Chem., Int. Ed. 2000, 39, 3490-3491. Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nature Biotech. 2000, 18, 1096-1100. Hall, D. B.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 5045-5046. J. AM. CHEM. SOC. 2002, 124, 12477-12485
9
12477
ARTICLES
Fahlman et al.
thought to base-stack continuously, which permits charge transfer through them. A large proportion of the studies on charge transfer through DNA have been carried out using DNA constructs containing covalently attached chemical groups for initiating charge transfer. However, a few direct measurements of charge transfer have also been made. Measurements made on ∼600 nm long “ropes” of intertwined, desiccated DNA,17 and later, through individual DNA duplexes,18 confirmed the conductive ability of DNA. To date, detailed (i.e., nucleotide-resolution) examinations of conduction through DNA still rely significantly on the use of covalently tethered photooxidant moieties, such as anthraquinone or rhodium- or ruthenium-containing organometallic complexes. Such strategies permit the use of custom-designed DNA sequences and the precise placement upon the DNA duplex of the photooxidant moiety, such that significant control can be exercised on where charge transfer is initiated. The different chemical groups that have been used as photooxidants, along with the interpretations obtained using such groups are summarized in various review articles.19-20 Fundamental examinations of the conductive properties of DNA have recently expanded into two related areas of interest. One is the use of DNA to template other conducting materials at the nanometer scale and the other is the examination of conduction through DNA structures more complex than the conventional duplex (double helix). Thus, DNA has been used to template the formation of silver wires21 and for the assembly of gold colloids.22 A novel metal-DNA complex, known as M-DNA, has also been reported to act as an efficient electrical conductor.23 Exploration of charge transfer in other DNA structures has included studies on DNA/RNA heteroduplexes,24-25 Z-form DNA,26 DNA triplexes,27-28 DNA guanine quartets,29 DNA aptamers,30 and immobile four-way DNA Holliday junctions (4W-junctions).31-32 In this paper, we have examined charge transfer in immobile DNA 4W-junctions (“single crossovers”) using two different appended photooxidant moieties, and, on the basis of the results obtained, we offer a reevaluation of results published by Barton and colleagues.32 Figure 1 illustrates schematically the structure of an immobile DNA 4W-junction. Four DNA oligonucleotides with precisely tailored base complementarities are used to assemble these junctions. Under low Mg2+ concentrations (180 Å.47,55 Our 30-base pair duplex only measured ∼100 Å from end to end, by comparison. A second set of comparisons could to be made between the AQ- and Rh-derivatized bulged duplexes (labeled as “dsDNAB” in Figure 4A,B). Guanine-damage patterns were somewhat different between the two: In the AQ-dsDNA-B, a reduction in cleavage levels was observed at the guanines distal (with respect to the site of AQ-modification) to the bulge (the position of the bulge is indicated by brackets in Figure 4. By contrast, the Rh-dsDNA-B duplexes exhibited a more uniform cleavage on either side of the bulge. In other words, the AQ-dsDNA-B appeared to be more responsive to the presence of the bulge as a disruption to base-stacking, and hence, to charge transfer. Comparison of guanine cleavage in the 4W-junction constructs highlights significant differences, depending on whether the DNA is derivatized with Rh or with AQ. With strand 4.1 (see Figure 4D) 32P-labeled at its 5′ end, cleavage was observed throughout the strand for both types of derivatizations (Figure 4A,B), whereas in experiments with strand 2.3 5′-labeled, high levels of cleavage were observed with the Rh-modified 4Wjunction (Figure 4A), but not with the AQ-modified 4W-junction (where a low degree of strand cleavage, barely detectable over those in the “dark”, nonirradiated controls were seensFigure 4B). To confirm that the charge migration in the AQ- modified 4W-junction remained localized to the coaxial stack of Stems I and IV, a second series of experiments were carried out. Photoirradiation experiments were conducted as described above, but with strand 3.4 5′-labeled with 32P. This strand bridges Stems III and IV; therefore, it could be predicted that piperidine-dependent cleavage would be observed only in the 3′ half of this sequence (when AQ was the photooxidant). Figure 4C shows the results of photoirradiation experiments on Rhand AQ-modified 4W-junctions when strand 3.4 was labeled. The Rh-modified construct exhibited cleavage at all guanines, whereas the AQ- modified sample only displayed cleavage at a guanine doublet located in Stem IV. (55) Henderson, P. T.; Jones, D.; Hampikian, G.; Kan, Y.; Schuster, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8353-8358. (56) Lilley, D. M. J. Quar. ReV. Biophys. 2000, 33, 109-159. J. AM. CHEM. SOC.
9
VOL. 124, NO. 42, 2002 12481
ARTICLES
Fahlman et al.
Figure 4. Photoirradiation of DNA assemblies. (A) Samples of gel-purified DNA constructs modified with Rh(phi)2(byp′)3+ were either irradiated at 366 nm for 210 min (hV lanes) at 6-10 °C, or left in the dark (D lanes). Lanes 4.1* and 2.3* show 4-way junction samples with their 4.1 and 2.3 strand, respectively, 5′-labeled with 32P. The dsDNA and dsDNA-B are samples, respectively, of duplex DNA and duplex DNA containing a bulge of two unpaired adenines. Following irradiation, all samples (including dark controls) where treated with hot piperidine (as described in the Materials and Methods), then loaded on a 12% sequencing gel along with the appropriate Maxam-Gilbert sequencing ladders. Arrows indicate the location of the junction on each sequence. (B) Identical experiments to that in (A), except that the DNA was modified with anthraquinone in place of Rh(phi)2(byp*)3+. (C) 4W-junctions modified with either anthraquinone or Rh(phi)2(byp*)3+, that were purified and treated as in (A) but possessed a 32P label on the 5′ end of strand 3.4. (D) Mapped sites of cleavage on the Rh(phi)2(byp*)3+ and anthraquinone modified 4-way junctions, respectively. Arrows indicate the relative intensities of piperidine-dependent cleavage at the indicated guanines.
Control experiments were carried out (under the same conditions as described above) with both the Rh- and the AQ4W-junction samples, such that 4W-junctions possessing tethered photooxidant but not possessing a 5′-32P label were photoirradiated mixed with 32P labeled 4W-junctions lacking the tethered photooxidant. In both the Rh- and the AQexperiments, no piperidine-dependent cleavage was observed upon photoirradiation (data not shown), suggesting that, at least under these conditions, intermolecular association of 4Wjunctions (involving, possibly, crossintercalation of the photooxidant moiety) was not a significant occurrence. The positions of cleavage and cleavage intensity on the Rhand AQ-modified 4W-junctions are summarized in Figure 4D, with arrow sizes reflecting the relative magnitudes of cleavage at each site. This summary clearly shows that the cleavage pattern of the AQ-modified 4W-junction better fits the accepted structural model for an immobile 4W-junction, in which pairs of stems coaxially stack, each with one preferred partner. In such a structure, charge would be expected to flow to/from the AQ- (or Rh-) derivatized Stem I to the preferred stacking partner (Stem II) but not to Stems III or IV. In theory, some electron transfer might occur to Stem IV, onto which Stem I might stack to a lower degree of preference; however, no charge transfer should be expected to Stem III, with which Stem I can never stack in the magnesium-folded form of the immobile 4Wjunction56 From this perspective, the results obtained with the Rh-modified 4W-junctions did not easily fit what was known about the structure and dynamics of 4W-junctions.56 Our Rhdependent results agree with the results published by Odom et al.32 on an Rh(phi)2(byp′)3+-derivatized 4W-junction (of unrelated DNA sequence), where significant amount of piperidine dependent cleavage was observed in all four arms of the 4Wjunction. 12482 J. AM. CHEM. SOC.
9
VOL. 124, NO. 42, 2002
We first wanted to confirm that the Rh(phi)2(byp*)3+ complex covalently attached to a 4W-junction molecule in fact intercalated only into that duplex stem (i.e., Stem I) of the 4W junction to which it was covalently tethered (as opposed to bridging the gap and intercalating into Stem II of the same 4W-junction molecule- - which would explain our observation of guanine damage in all four helical stems). To test this we used direct photolysis mapping, whereby photoirradiation with shorter wavelength UV light (∼310 nm) causes direct DNA-strand cleavage at the site of intercalation of these types of rhodium complexes.49 Samples of Rh(phi)2(byp*)3+-modified 4W-junctions (and the appropriate double-stranded controlssdsDNA) were prepared such that only one of the strands 2.3, 3.4, and 4.1 was 5′-labeled at a time with 32P. Double stranded controls (dsDNA) were also assembled in this way. Aliquots of each sample were photoirradiated at 310 nm for 45 min, and then immediately loaded onto a 10% sequencing gel alongside nonirradiated (“dark”) controls of the same complexes. Only the 4W-junction constructs with label on strand 4.1 exhibited cleavage at the most 3′ end of this sequence (data not shown), suggesting that the tethered Rh(phi)2(byp*)3+ complex was indeed only intercalating into Stem I. One caveat with the above experiment, using this particular 4W-junction (4W-junction I), was that any putative intercalation into Stem II would only be observed as cleavage of the 32P-labeled strand 2.1, at its extreme 5′ end, generating 1-3 nucleotide pieces that in general are difficult to detect and to resolve using denaturing gel electrophoresis. To overcome this problem, another Rh-derivatized 4Wjunction (“4W-junction II”) was examined. Figure 5A shows the sequence and the helix-stacking partners of this new junction, as well as the sequence of the double-stranded control for this junction. In 4W-junction II, the preferred stacking partners are Stems I and II, to form one coaxial stack, and Stems III and IV
Properties of DNA Holliday Junctions
Figure 5. (A) Sequences of 4W-junction-II and of its double-stranded control. Duplex stems of the 4W-junction are numbered sequentially I′-IV′. (B) Direct photolysis experiment on the 4W-junction-II. Samples of gel-purified DNA constructs derivatized with Rh(phi)2(byp*)3+ were either irradiated at 312 nm for 45 min (hV lanes) at 6-10 °C or left in the dark (D lanes). Samples were then loaded directly onto a 12% sequencing gel. Samples treated in this way include the duplex control as well as 4Wjunction samples with strand 2′.3′, 4′.1′, or 3′.4′ individually 5′- 32P-labeled. Brackets highlight areas of direct strand cleavage. (C) Charge-conduction related damage at the guanines of the 4W-junction-II, following irradiation at 366 nm and piperidine treatment. Samples of gel-purified DNA constructs modified with Rh(phi)2(byp′)3+ were either irradiated for 210 min (hV lanes) at 6-10 °C or left in the dark (D lanes). Lanes indicated as 2′.3′ and 4′.1′, are 4-way junction samples with the 2′.3′ and 4′.1′ strand, respectively, being 32P-end labeled. The lane indicated as h/c mix contains the unmodified 32P-labeled 4-way junction irradiated in the presence of nonradiolabeled, Rh-modified 4-way junction. Following irradiation all samples (including dark controls) were treated with hot piperidine (as described in the Materials and methods), then loaded on a 12% sequencing gel alongside appropriate Maxam-Gilbert sequencing ladders. Arrows indicate the location of the junction upon each sequence.
pairing to form the other.34 If it were possible for the tethered Rh(phi)2(byp*)3+ moiety to span the separation between spatially adjacent stems (such as between Stems I and IV) of this 4Wjunction, it might also intercalate into Stem IV, with consequent 310-nm induced direct photolysis both at the 5′ end of strand 4′.1′ and at the 3′ end of strand 3′.4′. However, the only direct cleavage observed with this 4W-junction construct was at the 3′ end of strand 4′.1′ (Figure 5B), indicating that, at least using this assay, the only substantial interactions of the Rh- moiety was with Stem I. However, when this 4W-junction, carrying the Rh(phi)2(byp*)3+ modification, was tested for charge transfer, via irradiation with 366-nm light, piperidine-sensitive guanine damage was found in all four helical arms as shown in Figure 5C (as with 4W-junction I, above). The above data report what appear to be contradictory observations on charge transfer through DNA 4W-junctions. On
ARTICLES
one hand, the anthraquinone (AQ-) modified 4W-junction yielded guanine-damage data consistent with our understanding of coaxial stacking in 4-W junctions (with the two coaxial stacks formed by a 4W-junction I electrically insulated from each other). On the other hand, experiments with two different Rh(phi)2(byp*)3+-modified 4W-junctions suggested the opposite, that charge transfer could happen with equal probability down all four helical arms. This latter result was, however, consistent with the data of Odom et al. (2001), who examined 4W-junctions derivatized with a closely related Rh-complex (see Figure 2c). To dissect the reason for this apparent discrepancy, we first examined whether the different photooxidant-derivatized DNA constructs were well-behaved, “monomeric” samples, using nondenaturing (native) polyacrylamide gel electrophoresis. Native gels, especially when run in the appropriate buffer, maintain the structural integrity of macromolecular complexes such as 4W-junctions. To ensure that gel-running buffer conditions resembled as closely as possible the DNA irradiation conditions, gels were run in the presence of 2 mM Mg2+. As seen in Figure 6A, a significant proportion of the Rh-derivatized duplex (lane 1), as well as 4W-junction (lane 5) migrate as retarded bands in the gel (shown with an asterisk), consistent with their having formed some kind of higher-order aggregates. By contrast, when 32P-labeled DNA 4W-junction (Junction I) lacking the Rh-derivatization was co-incubated with nonradiolabeled but Rh(phi)2(byp*)3+-derivatized 4W-junction, no slowmigrating band of presumed DNA aggregate was observed (lane 4). Controls with 32P-labeled 4W-junctions lacking in any derivatization or AQ-modified 4W-junctions (lane 6), gave no sign of aggregate formation. Interestingly, the AQ-derivatized duplexes (lane 2) and 4W-junction (lane 6) did not show this apparent aggregation. To ensure that this aggregation was not an isolated phenomenon with a particular batch of assembled DNA constructs (which, moreover, had been gel-purified after assembly), we again assembled our DNA constructs in conditions identical to those used by Odom et al.32 in their published work on 4Wjunctions. Following the cooling step to anneal the constituent DNA strands into the various constructs, an aliquot of each sample was removed for examination by nondenaturing electrophoresis, whose 32P autroradiograph is shown (Figure 6B upper). The remainder of the samples were then incubated for 2 h at 18 °C, and examined in a similar fashion (Figure 6B lower). This incubation time is on the time scale of photoirradiations used to detect long-range charge transport in our assay (180 min) as well as in experiments reported in an earlier study on 4W-junctions (120 min).32 In agreement with our earlier results, what was observed again was that in duplexes (lane 1) and in 4W-junctions (lane 5) that were derivatized with Rh(phi)2(byp*)3+, a species of lower electrophoretic mobility (shown with an asterisk) emerged in time-dependent fashion. In these lanes, individual duplex and 4W-junction molecules were labeled with both Rh(phi)2(byp*)3+ and 32P, so that the DNA molecules visible by autoradiography necessarily also had the Rh-derivatization. By contrast, when 32P-labeled DNA 4Wjunction (Junction I) lacking the Rh-derivatization was coincubated with nonradiolabeled but Rh(phi)2(byp*)3+-derivatized 4W-junction, no slow-migrating band of presumed DNA aggregate was observed (lane 4). Again, when 4W-junctions lacked J. AM. CHEM. SOC.
9
VOL. 124, NO. 42, 2002 12483
ARTICLES
Fahlman et al.
(25 mM final) and incubated at room temperature for >20 min prior to loading on a nondenaturing gel. Such a treatment, however, led to only a modest decrease in the amount of aggregate formed, suggesting that these complexes were stable even in the absence of magnesium. Interestingly, prior work using the Rh(phi)2(byp*)3+ photooxidant for long-range electrontransfer experiments had made the unusual observation of higher levels of damage at guanine doublets relatively distant from the tethered Rh-complex than at guanine doublets in closer proximity to the rhodium complex. This phenomenon, appeared, moreover, to occur independent of the ionic strength of the solution or of the presence or absence of Mg2+.57 Discussion
Figure 6. (A) Nondenaturing gel electrophoresis of derivatized DNA constructs. Lanes 1: Duplex DNA derivatized with Rh(phi)2(byp*)3+ (Rh-); Lanes 2: Duplex DNA derivatized with anthraquinone (AQ-). Lanes 3: A nonderivatized 4-way junction; Lanes 4: 32P-labeled, underivatized 4-way junction incubated with nonradiolabeled, Rh-derivatized 4-way junction. Lanes 5: Rh-modified 4-way junction; Lanes 6: AQ- modified 4-way junction. Lanes 7-9 show mixtures of the oligonucleotides used to assemble the 4-way junctions, showing one, two or three of the strands, respectively. All samples contained 0.5 µM total DNA, with the exception of lane 4, which contained 0.5 µM of each construct, and lanes 5d an d 6d, in which the DNA was diluted 2-fold in TNME buffer prior to incubation. Samples were incubated overnight at 12 °C before loading on an 8% nondenaturing gel (0.5× TB, 4 mM MgCl2, 0.1 mM EDTA). Horizontal lines indicate the tops of the gels (bottom of the samples loading wells) and the asterisks indicate the rhodium-specific aggregates. (B) Identical to (A) but all samples contained 2 µM total DNA (with the exception of lane 4, which contained 2 µM of each construct). Samples were incubated in 50 mM Tris-acetate (pH 7.7), 1 mM EDTA, and 10 mM Mg-acetate for 5 (upper gel) or 120 (lower gel) minutes at 18 °C before loading. The gels were run under identical time and running conditions to each other.
any derivatization (lane 3) or were AQ-derivatized (lane 6), there were likewise no signs of aggregate formation. In other words, any DNA construct participating, under these conditions, in aggregate-formation necessarily had to possess the Rh(phi)2(byp*)3+ derivatization. Conversely, the presence of the AQderivatization, or the lack of any such derivatization, did not support the formation of aggregates. One distinction between the results described above and what we observed in comparable experiments carried out with gel-purified constructs was that gel-purification gave rise to higher levels of aggregation, attributable to the long elution time of the DNA constructs from the gel. This high level of aggregation is likely the reason we observed no preference for apparent charge transfer through the different duplex stems of the 4W-junction constructs, whereas the previous work by Odom et al. did find some preference (despite significant cleavage observed in all four arms of their 4W-junction construct). To test for any role of magnesium in promoting the aggregate formation, samples containing the Rh-dependent aggregate were also treated with a 5-fold excess (over magnesium) of EDTA 12484 J. AM. CHEM. SOC.
9
VOL. 124, NO. 42, 2002
Our reexamination of photoinduced charge transfer in immobile DNA 4W-junctions found discrepant results, which were dependent upon the identity of the photooxidant. We have identified the likely reason for this discrepancy as being the formation of higher order aggregates of DNA constructs containing the Rh(phi)2(byp*)3+ modification. This was not an occurrence isolated to the 4W-junctions, for aggregation was also observed, under the same experimental conditions, with simple DNA duplexes derivatized with Rh(phi)2(byp*)3+. A key point about the formation of the aggregate structures was the requirement that all participating DNA construct molecules possess the Rh(phi)2(byp*)3+ modification (i.e., 4Wjunction molecules not derivatized with Rh did not participate in aggregate formation). This observation explains why control experiments that have traditionally been used32 to show that intermolecular interactions were not occurring in these systems failed to detect aggregation. In such traditional control experiments, DNA constructs end-labeled with 32P but not with a Rh complex were photoirradiated in a mixture with DNA constructs derivatized with the Rh complex but not with 32P. Such experiments typically revealed an absence of piperidine-sensitive guanine damage in 32P-labeled DNA strands, which was taken to mean that no significant intermolecular association between individual molecules of DNA construct was occurring. However, autoradiography revealed the fate only of the 32P-labeled constructs, which neither possessed the Rh-derivatization themselves nor participated in aggregation (via association with the Rh moiety of a Rh-derivatized but nonradiolabeled construct). Our redesign of this important control, using DNA constructs in which individual molecules were both 32P-labeled and Rhderivatized, suggests that intermolecular association (aggregation) is an important feature of constructs carrying the rhodium complex. The detailed structure and composition of the higher order aggregates that we have identified cannot be specified as yet, given that they may incorporate two, three, or more participating construct molecules. Figure 7 illustrates “minimal” models for these aggregates (in this figure, the duplex aggregates are hypothesized to contain more participating duplex molecules than those forming from the 4W-junction because the duplex aggregatessFigure 6 lowersexhibit even lower elecrophoretic mobility than the 4W-junction aggregates). In both the duplex and 4W-junction versions of the aggregation model, the loci of cross-interaction or -intercalation are arbitrarily placed within the DNA helices, on the presumption that preferred sites for (57) Williams, T. T.; Barton, J. K. J. Am. Chem. Soc. 2002, 124, 1840-1841.
Properties of DNA Holliday Junctions
Figure 7. Minimal models of low gel mobility products (presumed DNA aggregates) formed from samples derivatized with Rh(phi)2(byp*)3+. Cross intercalation by rhodium modifications are presumed to be random such that a multitude of individual species are obtained.
such cross-interaction may not exist. In fact, support for this supposition comes from data from an untethered rhodium complex (Rh(phi)2(byp)3+), which had previously been demonstrated to exhibit no sequence selectivity in its DNA binding.58 A relatively random dispersement of such cross-intercalation sites would result in sufficiently low levels of direct, 313-nm induced cleavage at defined sites within the DNA to escape detectionsas was found in our direct photolysis experiments, especially if one considers that not all of the constructs would be involved in such aggregate structures. We carried out a series of extended irradiation times for such experiments, to attempt to detect putatively preferred sites for such cross-intercalation, but extended irradiation at shorter wavelengths (313 nm) resulted in a variety of lesions and cross-links in the Rh-modified DNA constructs, that made it impossible to interpret the data (data not shown). It is noteworthy in this respect that charge-transfer experiments carried out with 4W-junction double-crossovers, using a Rh photooxidant,31 did report an insulation of the two sets of stacked helices from one another (such that charge transfer was restricted to that one helical stack to which the Rh-complex was attached and into which it was intramolecularly intercalated). The double-crossover construct is, however, a significantly more rigid structure than the simple 4W-junction (single crossovers), both in terms of its global structure (the spatial disposition of its helices) and its freedom of movement of helices away from the stack.59 Owing to topological constraints, it may (58) Pyle, A. M.; Long, E. C.; Barton, J. K. J. Am. Chem. Soc. 1989, 111, 45204522.
ARTICLES
be expected that the central helical portions of double-crossover constructs are less likely to unwind (helical unwinding is necessary to permit intercalation within a helix). It is therefore quite conceivable that intermolecular aggregation involving double crossovers, mediated by inter-construct intercalation events, may be energetically disfavored, relative to such events occurring in single crossovers (i.e., 4W-junctions). In summary, we offer here evidence and propose a testable hypothesis to explain the observed, photooxidant-specific, differences that have been reported in charge transfer-related processes in different DNA helical constructs. Further investigations into the salt- and temperature-dependence of aggregate formation by differently derivatized DNA constructs, as well as structural investigations thereof, will help to throw further light on the subject. Conclusions. We provide experimental data that may help to reconcile the discrepancies observed in charge-transfer processes in DNA constructs when they utilize Rh(phi)2(byp*)3+ versus anthraquinone as the covalently tethered photooxidant, under our experimental conditions. The formation of intermolecular aggregate structures, involving cross-construct intercalation of the photooxidant, likely results in guanine damage that is not a result of the anticipated intramolecular intercalation process. Experimental evidence from using anthraquinone as a photooxidant also suggest that immobile 4W-junctions do indeed form “insulated” coxial stacks, as one would predict from their known 3-dimensional structure, if charge transfer were proceeding strictly through the base stacks. More broadly, the findings in this paper may provide a basis for understanding the discrepant and apparently irreconcilable results reported on the ability of different sequences within DNA to conduct charge. Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). We are deeply grateful to Jay Cadieux and Peter Wilson for advice and assistance with the synthetic procedures. We also thank Ross Hill, Marcy Tracy, Peter Unrau, and the members of the Sen lab. R.P.F. was an NSERC Postgraduate Scholar during this work. D. S. is a Senior Scholar of the Michael Smith Foundation for Health Research. JA020495N (59) Li, X.; Yang, X.; Qi, J.; Seeman, N. C. J. Am. Chem. Soc. 1996, 118, 6131-6140.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 42, 2002 12485
